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ORIGINAL RESEARCH published: 05 July 2021 doi: 10.3389/fmicb.2021.712030

(S)-3-Hydroxybutyryl-CoA Dehydrogenase From the Autotrophic 3-Hydroxypropionate/ 4-Hydroxybutyrate Cycle in Nitrosopumilus maritimus

Li Liu1, Daniel M. Schubert2, Martin Könneke3,4 and Ivan A. Berg1*

1 Institute for Molecular Microbiology and Biotechnology, University of Münster, Münster, Germany, 2 Department of Microbiology, Faculty of Biology, University of Freiburg, Freiburg, Germany, 3 Marine Group, MARUM Center for Marine Environmental Sciences, University of Bremen, Bremen, Germany, 4 Benthic Microbiology, Institute for Chemistry and Biology of the Marine Environments, University of Oldenburg, Oldenburg, Germany

Ammonia-oxidizing archaea of the phylum Thaumarchaeota are among the most abundant that exert primary control of oceanic and soil nitrification and are responsible for a large part of dark ocean primary production. They assimilate inorganic via an energetically efficient version of the 3-hydroxypropionate/4- hydroxybutyrate cycle. In this cycle, acetyl-CoA is carboxylated to succinyl-CoA, which Edited by: is then converted to two acetyl-CoA molecules with 4-hydroxybutyrate as the key Johann Heider, University of Marburg, Germany intermediate. This conversion includes the (S)-3-hydroxybutyryl-CoA dehydrogenase Reviewed by: reaction. Here, we heterologously produced the protein Nmar_1028 catalyzing this Yejun Han, reaction in thaumarchaeon Nitrosopumilus maritimus, characterized it biochemically University of Chinese Academy of Sciences, China and performed its phylogenetic analysis. This NAD-dependent dehydrogenase is highly James F. Holden, active with its , (S)-3-hydroxybutyryl-CoA, and its low Km value suggests that University of Massachusetts Amherst, the protein is adapted to the functioning in the 3-hydroxypropionate/4-hydroxybutyrate United States cycle. Nmar_1028 is homologous to the dehydrogenase domain of crotonyl-CoA *Correspondence: Ivan A. Berg hydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase that is present in many Archaea. [email protected] Apparently, the loss of the dehydratase domain of the fusion protein in the course of evolution was accompanied by lateral gene transfer of 3-hydroxypropionyl-CoA Specialty section: This article was submitted to dehydratase/crotonyl-CoA hydratase from Bacteria. Although (S)-3-hydroxybutyryl- Microbial Physiology and , CoA dehydrogenase studied here is neither unique nor characteristic for the HP/HB a section of the journal Frontiers in Microbiology cycle, Nmar_1028 appears to be the only (S)-3-hydroxybutyryl-CoA dehydrogenase in Received: 19 May 2021 N. maritimus and is thus essential for the functioning of the 3-hydroxypropionate/4- Accepted: 11 June 2021 hydroxybutyrate cycle and for the biology of this important marine archaeon. Published: 05 July 2021 Keywords: autotrophy, 3-hydroxypropionate/4-hydroxybutyrate cycle, Nitrosopumilus maritimus, ammonia- Citation: oxidizing archaea, sedula, 3-hydroxybutyryl-CoA dehydrogenase Liu L, Schubert DM, Könneke M and Berg IA (2021) (S)-3-Hydroxybutyryl-CoA INTRODUCTION Dehydrogenase From the Autotrophic 3-Hydroxypropionate/ 4-Hydroxybutyrate Cycle Biological inorganic carbon fixation, the oldest and quantitatively most important biosynthetic in Nitrosopumilus maritimus. process in Nature, proceeds via at least seven natural pathways (Berg, 2011; Fuchs, 2011; Front. Microbiol. 12:712030. Sánchez-Andrea et al., 2020). Two of these pathways, the 3-hydroxypropionate/4-hydroxybutyrate doi: 10.3389/fmicb.2021.712030 (HP/HB) and the dicarboxylate/4-hydroxybutyrate (DC/HB) cycles were found only in Archaea

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(Berg et al., 2010a). In both of these cycles, acetyl-CoA a C-terminal enoyl-CoA hydratase domain and an N-terminal is carboxylated to succinyl-CoA, which is then reduced to dehydrogenase domain (Ramos-Vera et al., 2011; Flechsler two acetyl-CoA molecules, regenerating the inorganic carbon et al., 2021). This protein is widespread among Archaea and acceptor and forming the final of the cycle(s) was probably present in the last common ancestor of the (Figure 1). However, the carboxylation reactions are different. TACK-superphylum consisting of T haum-, A ig-, C ren-, and The carboxylases in the DC/HB cycle are -dependent Korarchaeota (Williams et al., 2017). also possess pyruvate synthase and phosphoenolpyruvate carboxylase (Huber a homologous crotonyl-CoA hydratase/(S)-3-hydroxybutyryl- et al., 2008; Ramos-Vera et al., 2009), while a promiscuous CoA dehydrogenase. Although this protein is present in acetyl-CoA/propionyl-CoA carboxylase catalyzes both acetyl- autotrophically grown Metallosphaera sedula (Sulfolobales) cells CoA and propionyl-CoA carboxylations in the HP/HB cycle (Ramos-Vera et al., 2011; Liu et al., 2021), it is responsible (Chuakrut et al., 2003; Hügler et al., 2003). In the latter case, a only for a minute flux in the crotonyl-CoA conversion to number of specific are responsible for the conversion acetoacetyl-CoA in this archaeon (Liu et al., 2020, 2021). In of malonyl-CoA, the product of the acetyl-CoA carboxylase contrast to the organisms using the DC/HB cycle, M. sedula and reaction, to propionyl-CoA, which is then carboxylated and other Sulfolobales possess stand-alone crotonyl-CoA hydratase isomerized to succinyl-CoA (Figure 1). Further metabolism and (S)-3-hydroxybutyryl-CoA dehydrogenase enzymes in the of succinyl-CoA is identical in both cycles. Succinyl-CoA HP/HB cycle (Liu et al., 2020). Their crotonyl-CoA hydratase is reduction leads to the formation of 4-hydroxybutyrate, which is a promiscuous protein that has also a high 3-hydroxypropionyl- converted to 4-hydroxybutyryl-CoA and dehydrated to crotonyl- CoA dehydratase activity and thus catalyzes two (de)hydratase CoA. Crotonyl-CoA conversion to two acetyl-CoA molecules reactions in the cycle (Liu et al., 2021). Thaumarchaeota proceeds through the reactions known from β-oxidation: also use a similar promiscuous to catalyze these two hydration of crotonyl-CoA to (S)-3-hydroxybutyryl-CoA, its reactions (Liu et al., 2021). Interestingly, crenarchaeal and oxidation to acetoacetyl-CoA and cleavage to two acetyl-CoA thaumarchaeal 3-hydroxypropionyl-CoA dehydratases/crotonyl- molecules (Figure 1). CoA hydratases are closely related to bacterial enoyl-CoA The DC/HB cycle functions in anaerobic of hydratases but were retrieved independently by the ancestors of the orders Desulfurococcales and Thermoproteales (Huber et al., Sulfolobales and AOA from different bacteria (Liu et al., 2021). 2008; Ramos-Vera et al., 2009; Berg et al., 2010b), while the Here, we characterize (S)-3-hydroxybutyryl-CoA dehydrogenase HP/HB cycle is present in aerobic Sulfolobales (Crenarchaeota) from the ammonia-oxidizing thaumarchaeon Nitrosopumilus and ammonia-oxidizing Thaumarchaeota (AOA) (Ishii et al., maritimus, compare it with the enzyme from M. sedula 1997; Berg et al., 2007, 2010a; Könneke et al., 2014). For both and with the crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA cycles, variants exist. Although it appears that the cycles in dehydrogenases, and discuss the enzymological differences in Thermoproteales and Desulfurococcales evolved from a common these conversions in course of the DC/HB and HP/HB cycles. ancestor, the representatives of these two orders use a number of phylogenetically unrelated enzymes (e.g., succinyl-CoA reductase, succinic semialdehyde reductase) (Huber et al., 2008; MATERIALS AND METHODS Ramos-Vera et al., 2009; Flechsler et al., 2021). For the HP/HB cycle, two fundamentally different variants function in Cren- and Materials in Thaumarchaeota. The thaumarchaeal variant uses ADP/Pi- Chemicals and biochemicals were obtained from AppliChem producing 3-hydroxypropionyl-CoA and 4-hydroxybutyryl-CoA (Darmstadt, Germany), Cell Signaling Technology (Frankfurt, synthetases, while non-homologous synthetases produce AMP Germany), Fluka (Neu-Ulm, Germany), IBA (Göttingen, and PPi in the crenarchaeal HP/HB cycle in the corresponding Germany), Merck (Darmstadt, Germany), Roth (Karlsruhe, reactions (Berg et al., 2007; Hawkins et al., 2013, 2014; Germany), Sigma-Aldrich (Deisenhofen, Germany), or VWR Könneke et al., 2014). The usage of ADP-producing synthetases (Darmstadt, Germany). Materials for cloning and expression makes the thaumarchaeal HP/HB cycle the energetically most were purchased from Bioline (London, United Kingdom), efficient aerobic autotrophic pathway (Könneke et al., 2014). MBI Fermentas (St. Leon-Rot, Germany), New England The characteristic enzymes of these two variants of the cycle Biolabs (Frankfurt, Germany), or Novagen (Schwalbach, are mostly phylogenetically unrelated, implying the involvement Germany). Materials and equipment for protein purification of convergent evolution in their emergence (Berg et al., 2007; were obtained from Cytiva (Freiburg, Germany), Macherey- Könneke et al., 2014; Otte et al., 2015). Nagel (Düren, Germany), Millipore (Eschborn, Germany), Both DC/HB and HP/HB cycles (and all their variants) Pall Corporation (Dreieich, Germany), or Thermo Scientific have in common that crotonyl-CoA is converted to acetoacetyl- (Rockford, United States). Primers were synthesized by CoA (Figure 1). The corresponding enzymes are widespread in Sigma-Aldrich (Steinheim, Germany). prokaryotes, being an interesting example of the involvement of convergent evolution and vertical gene transfer in the emergence Microbial Strains and Growth Conditions of different autotrophic pathways, and a possible role of lateral N. maritimus strain SCM1 was cultivated at 28◦C in 15-l HEPES- gene transfer in this process. In the DC/HB cycle, this conversion buffered medium (pH 7.5) as previously described (Martens- is catalyzed by a bifunctional crotonyl-CoA hydratase/(S)-3- Habbena et al., 2009). Cells were stored at −20◦C and used for hydroxybutyryl-CoA dehydrogenase consisting of two domains, DNA isolation. Escherichia coli strain TOP10 and E. coli strain

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FIGURE 1 | The N. maritimus 3-hydroxypropionate/4-hydroxybutyrate cycle (A) and the I. hospitalis dicarboxylate/4-hydroxybutyrate cycle (B) adapted from Berg et al.(2010b) and Könneke et al.(2014). Enzymes: 1, acetyl-CoA carboxylase; 2, malonyl-CoA reductase; 3, malonic semialdehyde reductase; 4, 3-hydroxypropionyl-CoA synthetase; 5, 3-hydroxypropionyl-CoA dehydratase; 6, acryloyl-CoA reductase; 7, propionyl-CoA carboxylase; 8, methylmalonyl-CoA epimerase; 9, methylmalonyl-CoA ; 10, succinyl-CoA reductase; 11, succinic semialdehyde reductase; 12, 4-hydroxybutyryl-CoA synthetase; 13, 4-hydroxybutyryl-CoA dehydratase; 14, crotonyl-CoA hydratase; 15, (S)-3-hydroxybutyryl-CoA dehydrogenase; 16, acetoacetyl-CoA β-ketothiolase; 17, pyruvate synthase; 18, PEP synthase; 19, PEP carboxylase; 20, malate dehydrogenase; 21, fumarate hydratase; 22, fumarate reductase; 23, succinyl-CoA synthetase. CoASH, ; PEP, phosphoenolpyruvate; Fd, ferredoxin; MV, methyl viologen.

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Rosetta 2 (DE3) (Merck, Darmstadt, Germany) were cultivated at Purification of Recombinant Protein ◦ 37 C in lysogeny broth (LB) medium. Nmar_1028 The heterologously produced histidine-tagged (his-tagged) CoA-Esters Synthesis Nmar_1028 was purified using affinity chromatography. The (S)- and (R)-3-hydroxybutyryl-CoA were synthesized from the supernatant was applied to 0.2-ml HisPur Ni-NTA Spin Column corresponding free acids by the mixed anhydride method (Thermo Fisher Scientific, Rockford, United States) that had (Stadtman, 1957) and then were purified using high-performance been equilibrated with 20 mM Tris-HCl containing 100 mM liquid chromatography (Zarzycki et al., 2009). Acetoacetyl-CoA KCl (pH 7.8). The unwanted proteins were washed out with and acetyl-CoA were chemically synthesized from diketene the same buffer containing 100 mM imidazole and the enzyme and acetic anhydride, respectively, as reported before (Simon was eluted with the same buffer containing 500 mM imidazole. ◦ and Shemin, 1953). 3-Hydroxypropionyl-CoA was synthesized The recombinant protein was stored in 50% glycerol at -20 C enzymatically with recombinant propionate CoA- after concentration using 10K Vivaspin Turbo 4 (Sartorius, from Clostridium propionicum (Selmer et al., 2002). Göttingen, Germany).

Gene Cloning Size Exclusion Chromatography Chromosomal DNA of N. maritimus was extracted using an The native molecular mass of Nmar_1028 was determined by illustra bacteria genomicPrep Mini Spin Kit (GE Healthcare). gel filtration on a Superdex 200 Increase column (24-ml volume; −1 The gene encoding (S)-3-hydroxybutyryl-CoA dehydrogenase Cytiva) at a flow rate of 0.75 ml min . The column was (Nmar_1028) was amplified by Q5 High-Fidelity DNA equilibrated with buffer containing 20 mM Tris-HCl (pH 7.8) Polymerase (NEB, Frankfurt, Germany) from genomic DNA of and 150 mM KCl. To make a standard curve, apoferritin (horse N. maritimus using the forward primer (50-GCG GCC ATA TCG spleen, 443 kDa), alcohol dehydrogenase (yeast, 150 kDa), bovine AAG GTC GTC ATA TGG CAG TAA AAA ATA TCA CAA serum albumin (BSA, 66 kDa), and carbonic anhydrase (bovine 0 erythrocytes, 29 kDa) were used. The eluted fractions were stored TTT TAG-3 ) introducing an NdeI restriction site (italicized) at ◦ the initiation codon and the reverse primer (50- TCT CAT GTT in 50% glycerol at -20 C. TGA CAG CTT ATC ATC GAT AAG CTT GAT TCA CTG CAT TGA TGT GAG-30) introducing a HindIII site (italicized) after Enzyme Assays the stop codon. Touchdown PCR was used as follows: initial All enzyme activities of Nmar_1028 were measured denaturation 3 min at 98◦C, 20 cycles of 30-s denaturation at spectrophotometrically in 300 µl reaction mixture at 30◦C at ◦ ◦ ◦ −1 −1 −1 −1 98 C, 30-s primer annealing at 57 C (gradually reduced 0.2 C 365 nm (εNADH = 3.4 mM cm , εNADPH = 3.5 mM cm ; per cycle) and 40-s elongation at 72◦C, followed by 15 cycles Bergmeyer, 1975). (S)-3-hydroxybutyryl-CoA dehydrogenase of 30-s denaturation at 98◦C, 30-s primer annealing at 53◦C activity was detected in 100 mM Tris-HCl (pH 7.8) containing and 40-s elongation at 72◦C. The PCR product was treated with 0.5 mM NAD(P), 0.2 mM (S)-3-hydroxybutyryl-CoA, and the corresponding restriction enzymes and then the gene was purified enzyme. The concentration of (S)-3-hydroxybutyryl- ligated into pET16b using T4 DNA (NEB). The cloning of CoA was varied (0.005–0.2 mM) for Km determination. Activity a gene into this vector with NdeI and HindIII restrictases adds of (S)-3-hydroxybutyryl-CoA dehydrogenase in the direction of an N-terminal His10-tag to the produced protein. The obtained NAD(P)H oxidation was measured in 100 mM Tris-HCl (pH plasmid was transformed into E. coli TOP10 for amplification. 7.8) containing 0.5 mM NAD(P)H, 0.2 mM acetoacetyl-CoA and purified enzyme. The concentration of acetoacetyl-CoA Heterologous Expression in E. coli was varied (0.005–0.2 mM) for Km determination. To illustrate the effect of pH on enzyme activity, 50 mM MOPS/50 mM The recombinant vector pET16b-Nmar_1028 was transformed Tris (pH 7.0, 7.4) and 100 mM Tris (7.8, 8.0, 8.6, and 9.0) were into E. coli Rosetta 2 (DE3). The cells were grown at 37◦C used. One enzyme unit (U) corresponds to 1 µmol substrate in LB medium with 100 µg ampicillin ml−1 and 34 µg converted per minute. chloramphenicol ml−1. Expression of Nmar_1028 was induced at optical density (OD578nm) of 0.6 with 0.2 mM isopropyl-β-D- thiogalactopyranoside (IPTG), and the temperature was lowered Database Search and Phylogenetic to 16◦C. The cells were harvested after additional growth for 15 h Analysis and stored at -20◦C until use. Query sequences for the database searches were obtained from NCBI data base. The BLASTP searches were performed via NCBI 1 Preparation of Cell Extracts BLAST server (Altschul et al., 1990). The phylogenetic tree was constructed by using the maximum likelihood method and Jones- Frozen cells were suspended in a double volume of 20 mM Taylor-Thornton (JTT) matrix-based model (Jones et al., 1992) Tris-HCl (pH 7.8) containing 0.1 mg ml−1 DNase I. The in MEGA-X (Kumar et al., 2018). 145 amino acid sequences cell suspension was sonicated for 60 cycles of 1-s pulse and were involved in this analysis. All positions containing gaps were 2-s pause, and the cell lysate was centrifuged for 20 min (14,000 rpm; 4◦C). The supernatant (cell extract) was used for protein purification immediately. 1http://www.ncbi.nlm.nih.gov/BLAST/

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completely deleted. The GenBank accession numbers for the protein sequences are listed in Supplementary Table 1. Other Methods DNA sequence determination of purified plasmids was performed by Eurofins (Ebersberg, Germany). Protein concentration was measured according to the Bradford method (Bradford, 1976) using BSA as a standard. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 12.5%) was performed as previously described (Laemmli, 1970). Proteins were visualized using Coomassie blue staining (Zehr et al., 1989). Protein identification was performed at the IZKF Core Unit Proteomics Münster based on tryptic in-gel digestion and mass spectrometric analysis using Synapt G2 Si coupled to M-Class (Waters Corp.). Km and Vmax values were calculated by GraphPad Prism5 software. FIGURE 2 | SDS-PAGE (12.5%) of fractions obtained during purification of heterologously produced Nmar_1028. Lane 1, molecular-weight markers (labeled in kDa); lane 2, cells before induction; lane 3, cells after induction; RESULTS lane 4, cell extract; lane 5, flowthrough from Ni-NTA column; lane 6, wash with 50 mM imidazole; lane 7, elution with 500 mM imidazole; lane 8, the first peak Characterization of Recombinant during gel filtration; lane 9, the second peak during gel filtration. Nmar_1028 Using a bioinformatic analysis, Nmar_1028 was identified as a putative (S)-3-hydroxybutyryl-CoA dehydrogenase in efficiency (kcat/Km) for NAD was 200 times higher than that N. maritimus (Könneke et al., 2014). Here, we amplified the gene for NADP (Table 1), being consistent with the published data nmar_1028, cloned it in pET16b vector (resulting in the vector for 3-hydroxybutyryl-CoA dehydrogenases. Apart from the pET16b-Nmar_1028), and heterologously expressed it in E. coli. Clostridium kluyveri enzyme (Madan et al., 1973), all other The recombinant protein was present in the soluble protein 3-hydroxybutyryl-CoA dehydrogenases characterized so far were fraction (Figure 2). The molecular weight of purified protein (best) active with NAD. Although the capability to use NADP as on SDS gel (45 kDa) was close to the predicted size (44.8 kDa). an alternative coenzyme was observed in some cases, the catalytic The identity of the obtained protein was confirmed using efficiency for this was very low (Osumi and Hashimoto, tryptic in-gel digestion and mass spectrometric analysis (PLGS 1980; Liu et al., 2020). score: 12489.25; peptides: 90; theoretical peptides: 40; coverage: The activity of Nmar_1028 was also measured in the reverse 98%; precursor RMS mass error: 10.1806 ppm; products: 1168; direction with NADH as acetoacetyl-CoA reduction to (S)-3- products RMS mass error: 10.8739 ppm; products RMS RT hydroxybutyryl-CoA (Table 1). The corresponding Vmax and −1 error: 0.01313549 min). Gel filtration chromatography showed Km values of the enzyme were 144.8 U mg protein and 26 the enzyme was eluted in two peaks. The molecular weight µM, respectively (with 0.5 mM NADH). The kcat/Km value for of these two peaks were estimated as 89.9 and 43.8 kDa, NADH was 5,271 s−1 mM−1, no activity was detected with 5 mM respectively, indicating that the enzyme was present in dimeric NADPH, revealing NADH as the electron donor for the enzyme. and monomeric forms in the preparation. The (S)-3-hydroxybutyryl-CoA dehydrogenase activity of The specific activity of the protein after His-tag purification Nmar_1028 was affected by changes in pH (Figure 3), gradually in (S)-3-hydroxybutyryl-CoA dehydrogenase reaction was increasing with increase of pH from 7 to 9 with ∼30% activity 36.2 ± 1.9 U mg−1 protein, and the corresponding activities for at pH 7.0 or 7.4 in comparison to 100% activity at pH 9. On the the dimeric and monomeric forms of enzyme were 4.6 ± 0.4 opposite, the activity for the reverse reaction was maximal in the U mg−1 protein and 69.6 ± 1.0 U mg−1 protein, respectively. buffers with lower pH (7.0–7.8), with remarkable activity decrease Therefore, the activity of the dimeric protein was only 6.6% with the pH change from 7.8 to 9.0 (Figure 3). These results are compared to the activity of the monomeric one. Only the typical for NAD-dependent dehydrogenases, as NAD+ reduction monomeric enzyme was used for the following measurements leads to the formation of NADH and H+. of the dehydrogenase activity (Table 1). The enzyme was As the Mycobacterium tuberculosis 3-hydroxybutyryl-CoA specific for NAD with high Vmax and low Km values for (S)- dehydrogenase activity was enhanced by divalent metals (Taylor 3-hydroxybutyryl-CoA (98.6 U mg−1 protein and 19 µM, et al., 2010), we performed the corresponding assays with the respectively, as measured with 2 mM NAD). The activity with N. maritimus protein. Nmar_1028 did not require addition of 0.5 mM NAD was slightly lower (Table 1). Only low activity divalent metals in the reaction mixture for activity (Table 2). with 3-hydroxypropionyl-CoA was detected (0.02 U mg−1 The incubation of the enzyme in the presence of divalent metals protein), confirming high specificity of the enzyme toward resulted rather in inhibition of its activity. Whereas Mg2+, Mn2+, (S)-3-hydroxybutyryl-CoA. Besides NAD, the enzyme is able to Ni2+, and Co2+ had only subtle effect on the activity, the addition use NADP as an electron acceptor. Nevertheless, its catalytic of Fe2+ and Zn2+ strongly affected the Nmar_1028 activity.

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TABLE 1 | Catalytic properties of heterologously produced (S)-3-hydroxybutyryl-CoA dehydrogenase Nmar_1028.

−1 −1 −1 Substrate Vmax (U mg protein) Km (mM) kcat/Km (s mM )

Forward reaction a (S)-3-Hydroxybutyryl-CoA (with 2 mM NAD) 98.6 ± 3.3 0.019 ± 0.002 3877 (S)-3-Hydroxybutyryl-CoA (with 0.5 mM NAD) 76.1 ± 3.4 0.017 ± 0.003 3344 NAD (with 0.2 mM (S)-3-hydroxybutyryl-CoA) 84.4 ± 2.8 0.10 ± 0.02 630 NADP (with 0.2 mM (S)-3-hydroxybutyryl-CoA) 10.1 ± 0.9 2.92 ± 0.63 3 Reverse reaction b Acetoacetyl-CoA (with 0.5 mM NADH) 144.8 ± 7.7 0.026 ± 0.004 4045 NADH (with 0.2 mM acetoacetyl-CoA) 112.9 ± 7.3 0.016 ± 0.006 5271

aNo activity was detected with (R)-3-hydroxybutyryl-CoA (detection limit ≤ 0.012 U mg−1). The specific activity was 0.02 U mg−1 protein with 3-hydroxypropionyl-CoA. b≤ 0.006 U mg−1 protein with 5 mM NADPH.

FIGURE 3 | Effect of pH on the forward (A) and reverse (B) reaction rates of recombinant (S)-3-hydroxybutyryl-CoA dehydrogenase Nmar_1028. (A) 100% activity corresponds to 69.6 ± 2.7 U mg-1 protein measured with 0.2 mM (S)-3-hydroxybutyryl-CoA and 0.5 mM NAD+ at pH 7.8. (B) 100% activity corresponds to 54.9 ± 7.2 U mg-1 protein measured with 0.2 mM acetoacetyl-CoA and 0.5 mM NADH at pH 7.8. Values are means ± standard deviations of results from three independent measurements.

TABLE 2 | Influence of divalent cations on the activity of recombinant to that of Nmar_1028, while its Km value is 6 times higher (S)-3-hydroxybutyryl-CoA dehydrogenase Nmar_1028. than that of Nmar_1028 (Tables 1, 3). Msed_1423 is the Substrate Relative activity (%) main (S)-3-hydroxybutyryl-CoA dehydrogenase in M. sedula −1 with the highest Vmax value (96 U mg protein) and the No addition 100a lowest Km value (5 µM) to its substrate (Table 3). The EDTA (0.5 mM) 100 ± 1 catalytic properties of Nmar_1028 are rather comparable MgCl2 (5 mM) 90 ± 10 with those of Msed_1423 than of Msed_0399. Indeed, the MnCl (5 mM) 88 ± 2 2 kcat/Km values of Nmar_1028 and Msed_1423 were 5- and NiCl2 (1 mM) 79 ± 8 20-fold higher than the corresponding value of Msed_0399 CoCl2 (1 mM) 76 ± 5 in the (S)-3-hydroxybutyryl-CoA dehydrogenase reaction FeCl2 (1 mM) 35 ± 4 (Tables 1, 3). ZnCl2 (1 mM) 1.5 ± 0.3 To trace the evolution of the HP/HB cycle, we a100% activity corresponds to 69.6 ± 1.4 U mg−1 protein measured with 0.2 mM performed a comparative phylogenetic analysis (Figure 4) (S)-3-hydroxybutyryl-CoA and 0.5 mM NAD. of the corresponding enzymes. The homologs of (S)- 3-hydroxybutyryl-CoA dehydrogenase Nmar_1028 Distribution and Phylogenetic Analysis of were found in the genomes of all sequenced AOA, suggesting that all these archaea share the ability to grow N. maritimus (S)-3-Hydroxybutyryl-CoA autotrophically by using the HP/HB cycle. Moreover, Dehydrogenase these homologs formed a separate cluster containing the Whereas Nmar_1028 is the only homolog for (S)-3- sequences of AOA, which is phylogenetically separated hydroxybutyryl-CoA dehydrogenase in the genome of from the cluster of the corresponding crenarchaeal N. maritimus, several phylogenetically related proteins can sequences (Figure 4). Interestingly, Nmar_1028 and catalyze this reaction in M. sedula (Liu et al., 2020). Msed_0389 Msed_1423 share 42/60% of sequence identity/similarity, has only low (S)-3-hydroxybutyryl-CoA dehydrogenase activity. whereas Nmar_1028 and the dehydrogenase domain of The activity of bifunctional crotonyl-CoA hydratase/(S)-3- Msed_0399 share 41/62% of sequence identity/similarity hydroxybutyryl-CoA dehydrogenase Msed_0399 is comparable (according to the BLASTP search). It appears that

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TABLE 3 | Catalytic properties of various so-far-characterized 3-hydroxyacyl-CoA dehydrogenases toward 3-hydroxybutyryl-CoA and/or acetoacetyl-CoA.

Organism Protein Gene 3-Hydroxybutyryl-CoA Acetoacetyl-CoA References

Vmax/kcat Km kcat/Km Vmax/kcat Km kcat/Km

Nitrosopumilus HBDH Nmar_1028 98.6/74 0.019 3,877 144.8/108 0.026 4,160 This work maritimus SCM1 (2 mM (2 mM (2 mM NAD) NAD) NAD) 76.1/57 0.017 3,344 (0.5 mM (0.5 mM (0.5 mM NAD) NAD) NAD) Metallosphaera HBDH Msed_1423 96/75(0.5 mM 0.005 14,957 NR NR NR Liu et al., 2020 sedula DSM 5348 NAD) CCH/HBDH Msed_0399 76/90a 0.12 748 NR NR NR Ramos-Vera et al., 22.6/19a 0.06 445 2011; Hawkins 27.6/33a 0.2 163 et al., 2014; Liu et al., 2020 Ignicoccus CCH/HBDH Igni_1058 117/152 0.086 1,800 NR NR NR Flechsler et al., hospitalis 2021 Cupriavidus HBDH/enoyl- H16_A0461 NR NR NR 149/216 0.048 4,500 Volodina and necator H16 CoA Steinbüchel, 2014 hydrataseb HBDH RePaaH1 NR NR NR 85.4/46 0.018 2,530 Kim J. et al., 2014 Clostridium HBDH CbHBD NR NR NR 56.8/29 0.028 1,035 Kim E. J. et al., butyricum 2014 Caenorhabditis HADH cHAD NR NR NR 78/45 0.072 600 Xu et al., 2014 elegans Bristol N2 Mycobacterium HBDH FadB2 0.19/0.096 0.0435 2 0.13/0.065 0.0656 1 Taylor et al., 2010 tuberculosis H37Rv Escherichia coli Fatty acid ? NR NR NR 64/288 0.066 4,364 Binstock and ATCC 11775 oxidation Schulz, 1981 complex Mycolicibacterium HADH ? NR NR NR 12.5/10 0.036 291 Shimakata et al., smegmatis ATCC 1979 14468 Homo sapiens Short-chain SCHAD I NR/119 0.0225 5,290 NR/1,287 0.0166 77,500 Filling et al., 2008 HADH I Short-chain SCHAD II NR/4.83 0.0852 58 NR/23.8 0.0257 927 Filling et al., 2008 HADH II Short chain rSCHADH6 NR NR NR 459/252 0.0187 13,500 Barycki et al., 1999 L-HADH Short chain SCHAD NR/0.011 0.043 0.26 NR NR NR He et al., 1999 L-HADH L-HADH HBHAD NR NR NR NR/37 0.089 416 He et al., 1998 Rattus norvegicus HADH HAD NR NR NR 976/569 0.044 13,000 Liu et al., 2004 HADH I ? 270/144 0.0699 2,060 820/437 0.0169 25,878 Osumi and Hashimoto, 1980 HADH II ? 15.6/20 0.0422 474 23.5/30 0.0397 760 Osumi and Hashimoto, 1980 Sus scrofa L-HADH HAD NR NR NR NR/330 0.003 110,000 He and Yang, 1998 L-HADH ? 39/21 0.0072 2,889 230/123 0.0118 10,395 He et al., 1989 L-HADH ? NR NR NR 426/227 0.06 3,787 Noyes and Bradshaw, 1973

−1 −1 Vmax, U mg protein; kcat, s ;Km, mM. (S)-3-hydroxybutyryl-CoA dehydrogenase, HBDH; crotonyl-CoA hydratase, CCH; 3-hydroxyacyl-CoA dehydrogenase, HADH?, a ◦ ◦ b unknown; NR, not reported. The Vmax values are normalized to 75 C based on the assumption that a 10 C rise in temperature doubles the reaction rate. Specific activity with crotonyl-CoA was 98 U mg−1.

despite their homology, these three proteins evolved DISCUSSION independently from each other, confirming the proposed convergent evolution of HP/HB cycle in Cren- Aerobic ammonia oxidation is a crucial part of the modern and Thaumarchaeota. cycle and an environmentally important process that

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FIGURE 4 | Phylogenetic tree of (S)-3-hydroxybutyryl-CoA dehydrogenase Nmar_1028. The sequences are marked as following: purple triangle, Nmar_1028; red triangle, Msed_1423; red star, Msed_0399; olive star, Tneu_0541 (from Pyrobaculum neutrophilum); turquoise star, Igni_1058 (from Ignicoccus hospitalis); , dehydrogenase; without , fusion protein; 80 sequences from TACK group in red, 15 sequences from non-AOA Thaumarchaeota in fuchsia, 49 sequences from Euryarchaeota in blue, 1 sequence from bacteria in green. The scale bar represents a difference of 0.1 substitutions per site. The percentage bootstrap values for the clade of this group were calculated in 1000 replications. Only values above 70% are shown (as ). The accession numbers are listed in Supplementary Table 1. The phylogenetic tree generated by maximum-parsimony algorithm (data not shown) was similar with minor exceptions.

is responsible for a large part of dark ocean inorganic carbon The HP/HB cycle exists in two variants, and the apparent fixation (Meador et al., 2020). This process is usually dated similarity in the intermediates of the cycle in AOA and back to the great oxidation event (Ren et al., 2019), and its in Sulfolobales is in sharp contrast with the results of the obligate requirement for oxygen can be accounted for by the biochemical and/or phylogenetic analysis of the corresponding fact that the first reaction in ammonia oxidation is performed enzymes (Könneke et al., 2014; Otte et al., 2015; Liu et al., via ammonia monooxygenase. It can be found only in a 2021). The difference in the autotrophic pathways in these limited number of bacteria and archaea. A recent phylogenomic two archaeal groups goes along with the difference in their analysis suggested that AOA originated from the terrestrial non- ecology and environmental requirements. While Sulfolobales ammonia-oxidizing ancestors and expanded to the shallow and like M. sedula gain energy from the highly exergonic aerobic then deep ocean upon their oxygenation (Ren et al., 2019), while hydrogen oxidation, AOA use a much less energetically favorable ammonia-oxidizing bacteria are much younger (Ward et al., reaction, the oxidation of ammonia to nitrite. Moreover, some 2021). Note however that this model is still under discussion AOA are adapted to perform near maximum autotrophic (Abby et al., 2020). The absence of the key genes of the growth at nanomolar concentrations of ammonia (Martens- HP/HB cycle in the non-ammonia-oxidizing Thaumarchaeota Habbena et al., 2009), further highlighting the high efficiency (non-AOA) and their presence in all AOA studied so far (Ren of their metabolism. Correspondingly, AOA and Sulfolobales et al., 2019) suggests that the thaumarchaeal HP/HB cycle convergently evolved the pathways that are best adapted to their evolved in AOA ancestors de novo, and that the last common needs, with a less energetically efficient but kinetically superior ancestor of AOA had already a fully evolved HP/HB cycle. variant functioning in Sulfolobales (Könneke et al., 2014). Our analysis also confirms this model, showing the (S)-3- (S)-3-Hydroxybutyryl-CoA dehydrogenases in M. sedula and hydroxybutyryl-CoA dehydrogenase studied here is present in N. maritimus are both homologous to the dehydrogenase all studied AOA, but not in non-AOA (Figure 4). Similarly, domain of crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA a 3-hydroxypropionyl-CoA dehydratase/crotonyl-CoA hydratase dehydrogenase (though to different clusters in the tree, Figure 4). gene was found only in ammonia-oxidizing representatives of the This fusion protein is present in many bacteria and archaea phylum Thaumarchaeota (Liu et al., 2021). and was predicted to be present in the common ancestor of

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Cren- and Thaumarchaeota (Williams et al., 2017). Apparently, archaeon. Moreover, some autotrophic Sulfolobales also have the protein lost its dehydratase domain in both archaeal only one enzyme to catalyze this conversion (Liu et al., groups in the course of evolution, probably because of 2020), what makes it as essential for the functioning of the its redundancy. Indeed, the HP/HB cycle possesses two cycle as any other enzyme involved in the cycle. Apart from very similar (de)hydratase reactions, crotonyl-CoA hydratase autotrophic CO2 fixation in the HP/HB and DC/HB cycles, and 3-hydroxypropionyl-CoA dehydratase. To catalyze these the enzyme participates in various metabolic process like β- reactions, both AOA and Sulfolobales possess a promiscuous oxidation, polyhydroxyalkanoate metabolism, or fermentations. 3-hydroxypropionyl-CoA dehydratase/crotonyl-CoA hydratase The low Km values of N. maritimus and M. sedula enzymes that was laterally transferred from bacteria to their ancestors to (S)-3-hydroxybutyryl-CoA suggest their adaptation to the (Liu et al., 2021). The importance of the acquisition of bacterial functioning in the HP/HB cycle. Moreover, these enzymes genes for the evolution of AOA has been shown earlier are of biotechnological interest and could be applied for the (Deschamps et al., 2014). biosynthesis of e.g., n-butanol (Kim and Kim, 2014). The HP/HB The apparent independent loss of the enoyl-CoA hydratase cycle is a promising candidate for microbial production of domain in the ancestors of AOA and Sulfolobales suggests that chemicals from CO2 (Loder et al., 2016), and the studied the separation of the hydratase and dehydrogenase reactions enzyme can be used e.g., to the reconstitution of the HP/HB should give certain advantages for organisms using the HP/HB cycle or similar process in a proper mesophilic host cycle. What could it be? The crotonyl-CoA hydratase/(S)-3- (Fuchs and Berg, 2014). hydroxybutyryl-CoA dehydrogenase Msed_0399 is only weakly active with 3-hydroxypropionyl-CoA. We suggest that an acquisition of a promiscuous bacterial hydratase capable of DATA AVAILABILITY STATEMENT catalyzing both of these reactions with high efficiency made the dehydratase domain of a fusion protein redundant, leading The original contributions presented in the study are included to its loss. In addition, the analysis of catalytic properties in the article/Supplementary Material, further inquiries can be of different (S)-3-hydroxybutyryl-CoA dehydrogenases suggests directed to the corresponding author/s. that the fusion proteins have much higher Km values to (S)- 3-hydroxybutyryl-CoA than the stand-alone dehydrogenases (Table 3). In fact, a two−step reaction catalyzed by a fusion AUTHOR CONTRIBUTIONS enzyme usually demonstrates an improved catalytic efficiency, which is usually attributed to a substrate channeling, however IB designed the experiments. LL, DS, and MK performed only if the intermediate concentration remains low (Rabe et al., the experiments. LL, DS, and IB analyzed the data. LL and IB wrote the manuscript. All authors read and approved the 2017). As the Km value to (S)-3-hydroxybutyryl-CoA in the dehydrogenase reaction is relatively high, this factor is probably final manuscript. not relevant for the functioning of the HP/HB cycle, and the merging of the dehydrogenase and hydratase in a fusion protein FUNDING for the HP/HB cycle becomes dispensable. Interestingly, autotrophic Crenarchaeota of the orders This work was funded by the Deutsche Forschungsgemeinschaft Thermoproteales and Desulfurococcales use the fusion protein to (BE 4822/5-1) and by China Scholarship Council catalyze the crotonyl-CoA hydratase and (S)-3-hydroxybutyryl- (201504910718, to LL). CoA dehydrogenase reactions in the DC/HB cycle (Huber et al., 2008; Ramos-Vera et al., 2009; Flechsler et al., 2021). This cycle does not require a 3-hydroxypropionyl-CoA dehydratase ACKNOWLEDGMENTS reaction. Furthermore, the crotonyl-CoA hydratase activity of the fusion protein is high (Ramos-Vera et al., 2009; Hawkins We thank S. König (IZKF Core Unit Proteomics, University of et al., 2014; Flechsler et al., 2021), making the tinkering around Münster, Münster) for the protein identification. this reaction unnecessary. The reaction studied here is neither unique nor characteristic for the HP/HB cycle. Nevertheless, the enzymatic conversion SUPPLEMENTARY MATERIAL of (S)-3-hydroxybutyryl-CoA to acetoacetyl-CoA is essential for the functioning of the cycle. The genome of N. maritimus The Supplementary Material for this article can be found is streamlined, and Nmar_1028 appears to be the only (S)-3- online at: https://www.frontiersin.org/articles/10.3389/fmicb. hydroxybutyryl-CoA dehydrogenase in this important marine 2021.712030/full#supplementary-material

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